Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice.

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.

Echoviruses 1 and 8 are closely related genetically, and bind to similar determinants within the VLA-2 I domain.

Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.

Multiple regions within the coxsackievirus and adenovirus receptor cytoplasmic domain are required for basolateral sorting.

The coxsackievirus and adenovirus receptor (CAR) mediates attachment and infection by coxsackie B viruses and many adenoviruses. In human airway epithelia, as well as in transfected Madin-Darby canine kidney cells, CAR is expressed exclusively on the basolateral surface. Variants of CAR that lack the cytoplasmic domain or are attached to the cell membrane by a glycosylphosphatidylinositol anchor are expressed on both the apical and basolateral surfaces. We have examined the localization of CAR variants with progressive truncations of the cytoplasmic domain, as well as with mutations that ablate a potential PDZ (PSD95/dlg/ZO-1) interaction motif and a putative tyrosine-based sorting signal. In addition, we have examined the targeting of two murine CAR isoforms, with different C-terminal sequences. The results suggest that multiple regions within the CAR cytoplasmic domain contain information that is necessary for basolateral targeting.

Survival, lung injury, and lung protein nitration in heterozygous MnSOD knockout mice in hyperoxia.

This study tested whether a strain of heterozygous Mn superoxide dismutase (SOD) knockout mice differed from wild types in response to lethal (100 or 85%) or sublethal (50 or 75%) oxygen exposures. Lung MnSOD activity was significantly (-40%) less in the heterozygous mice, and lung catalase activity was also significantly decreased. Total SOD activity, glutathione peroxidase, and glutathione reductase did not differ between heterozygous (+/-) and wild-type (+/+) mice. We exposed both heterozygous and wild-type mice to hyperoxia (50, 75, 85, or 100% oxygen) until death or for 48 hours to assess sublethal lung injury. Survival of the heterozygous and wild-type mice did not differ significantly in 100 or 85% oxygen. No mice of either genotype died in 50 or 75% oxygen (14-day exposures). Hyperoxia exposures significantly increased (by two-way ANOVA) the alveolar lavage protein concentration, percent neutrophils, and lung wet-dry/dry weight ratios. No significant differences occurred between the heterozygous and wild-type mice for any marker of injury at any oxygen level. Lavage fluid total nitrite concentrations did not differ at any oxygen level. Hyperoxia caused a similar degree of nitration of lung structural proteins detected by immunohistochemistry in both groups.

Hypoxic modulation of manganese superoxide dismutase promoter activity and gene expression in lung epithelial cells.

We investigated the effects of hypoxia (< 2.5% O2) on rat manganese superoxide dismutase (MnSOD) gene promoter-luciferase reporter constructs in transiently transfected lung epithelial cells (A549, L2, and E1A-T2) and fibroblasts (R9Ab). We cloned MnSOD promoter-luciferase reporter constructs (numbers refer to length in base pairs [bp] in the 5' direction from the transcription initiation site): 2,505, 1,064, 507, 405, and 289 into pGL2-Basic, a promoterless, firefly luciferase vector. Lung cells were transfected with MnSOD promoter-reporter constructs with or without thymidine kinase-driven Renilla luciferase (pRL-TK), and were exposed to air/5% CO2 or hypoxia (2.5% O2/5% CO2/balance N2) for 24 h. Hypoxia caused a significant (by two-way analysis of variance) consistent increase in luciferase in the A549 cell (human lung carcinoma) line. Greatest expression (> 3-fold increase) in hypoxia was associated with the 2,505-bp MnSOD promoter (normalized to cellular protein). Azide (10 microM) did not increase expression of the MnSOD reporter constructs. The 289-bp promoter was sufficient to express the reporter in air and to increase its expression in hypoxia. Promoter activity of the rat MnSOD 5' region, assessed by luciferase reporter constructs in A549 cells, increased in hypoxia. The increase was exclusive to A549 cells and did not occur in other cells.

Analysis of mouse intron 7 DNA sequence of the APP gene: comparison with the human homologue.

Mutations in the beta-amyloid precursor protein gene (APP) cause Alzheimer disease (AD) in certain families. The mature protein (APP) exists in several different isoforms resulting from alternative splicing of the primary transcript. Several lines of evidence indicate that particular isoform(s) of APP may contribute to the etiology of AD. One of the isoforms, APP695, lacks the Kunitz protease inhibitor (KPI) domain encoded by exon 7. APP695 is expressed predominantly in neurons, whereas the KPI domain containing isoforms, APP751 and APP770, are expressed ubiquitously. The ratio of APP751/APP695 mRNA tends to increase in the brain of AD patients. Furthermore, this ratio in mouse brain is much lower than that in human brain, and mice are resistant to the spontaneous development of beta-amyloidosis. In addition, transgenic mice that develop pathological changes similar to those of AD expressed more KPI-domain containing APP mRNA than transgenic mice without the changes. Previous studies imply that the controlling elements exist in the flanking sequences of the alternatively-spliced exons. Therefore, we have determined the DNA sequences of intron 7 and made a comparison between mouse and human DNA sequences of intron 7. Mouse intron 7 shares about 50% sequence identity with the human homologue, with higher sequence identity (approximately 85%) mainly in the 5' end (approximately 250 bp) of the intron. A palindromic sequence was found in both human and mouse intron 7 and showed subtle differences in their structure between the two species. Whether this sequence plays any roles in regulating alternative splicing of exon 7 remains to be determined. Human intron 7 contains a Alu element, which possesses potential retinoic acid and thyroid hormone responsive elements that might be involved in the regulation of alternative splicing. Mouse intron 7 sequence also contains a few repeat sequences which are specific to the genome of mice and rats. Homologies shared between human and mouse intron 7 sequences may contribute to the common characteristics of neuron-specific splicing of APP in both species. The unique features of the intron may account for differences between human and mouse brain in fine tuning of alternative splicing of the APP transcript, which may lead to their different susceptibilities to beta-amyloidosis.

Polymorphic tetranucleotide repeat site within intron 7 of the beta-amyloid precursor protein gene and its lack of association with Alzheimer's disease.

Mutations found in the beta-amyloid precursor protein (APP) gene in a small subset of patients with Alzheimer's disease (AD) are associated with the development of the disease. Several lines of evidence indicate that specific isoforms of APP generated by alternative splicing of the primary transcript may contribute to the etiology of AD. One of the isoforms, APP695, lacks the Kunitz protease inhibitor (KPI) domain and is produced predominantly in neurons by skipping exon 7 of the APP gene. Previous studies imply that the controlling elements for exon 7 skipping exist in the flanking sequences of the exon. Therefore, we have sequenced the human intron 7 of the APP gene and found a polymorphic tetranucleotide (ATTT)n repeat site within the intron 7. In 183 genetically unrelated subjects (97 AD patients and 86 controls), we found four alleles by polymerase chain reaction (PCR) amplification of the repeat site. Although no particular alleles are associated with AD, this newly identified polymorphic site may be useful in other genetic analyses since preliminary evidence suggests allele frequency differences between African Americans and Caucasians.

Expression of apolipoprotein E inhibits aggregation of the C-terminal fragments of beta-amyloid precursor protein.

An important role of apolipoprotein E in the amyloidogenesis of Alzheimer's disease is suggested by an accumulation of apolipoprotein E in beta-amyloid plaques and a genetic association between Alzheimer's disease and one of the allelic variants (APOE4) of apolipoprotein E. Overexpression of a C-terminal region of beta-amyloid precursor protein brings about aggregation of the C-terminal fragments in COS cells. This COS cell culture system was used to study effects of apolipoprotein E on aggregation of the C-terminal fragments. When both apolipoprotein E and the C-terminal fragments were overexpressed in COS cells, Western blot analyses revealed significant inhibition of aggregation of the C-terminal fragments. No significant differences between apolipoprotein E3 and E4 in the inhibitory activities were found by this method. Apolipoprotein E may inhibit formation of amyloid fibrils.

Overexpressions of cDNAs for beta-amyloid precursor proteins 695, 751, and 770 enhance the secretion of beta-amyloid precursor protein derivatives and the survival of P19-derived neurons.

P19 is a C3H mouse-derived line of multipotent embryonic carcinoma cells that differentiate into neural cells. P19 cell clones overexpressing the three major forms of beta-amyloid precursor protein from their cDNA constructs were established. Unlike a previous study in which P19-derived neurons had a limited alpha-secretase activity, all of these clones produced significant amounts of secreted beta-amyloid precursor protein. When treated with retinoic acid, these transformed lines differentiated into neurons and survived better than did nontransformed parental P19 cells. Furthermore, P19-derived neurons survived better in medium conditioned by the transformed P19 line, and survival was reduced by immunoabsorption with an antibody to beta-amyloid precursor protein. These results suggest neurotrophic effects of secreted beta-amyloid precursor protein and contrast with a previous report in which overexpression of a full-length cDNA for beta-amyloid precursor protein led to degeneration of P19-derived neurons. Western blot analysis suggested that this difference might result from different levels of expression of putative neurotoxic C-terminal fragments of beta-amyloid precursor protein; moreover, P19-derived neurons differ from P19 stem cells in the processing of these C-terminal fragments.